Biomedical subjects

U Schindler

Publications and source records attributed to U Schindler.

At least 19 recordsLinked to original sources

PubMed1998

Stat6 inhibits human interleukin-4 promoter activity in T cells.

The differentiation of naive T-helper (Th) cells into cytokine-secreting effector Th cells requires exposure to multiple signals, including exogenous cytokines. Interleukin-4 (IL-4) plays a major role in this process by promoting the differentiation of IL-4-secreting Th2 cells. In Th2 cells, IL-4 gene expression is tightly controlled at the level of transcription by the coordinated binding of multiple transcription factors to regulatory elements in the proximal promoter region. Nuclear factor of activated T cell (NFAT) family members play a critical role in regulating IL-4 transcription and interact with up to five sequences (termed P0 through P4) in the IL-4 promoter. The molecular mechanisms by which IL-4 induces expression of the IL-4 gene are not known, although the IL-4-activated transcription factor signal transducer and activator of transcription 6 (Stat6) is required for this effect. We report here that Stat6 interacts with three binding sites in the human IL-4 promoter by electrophoretic mobility shift assays. These sites overlap the P1, P2, and P4 NFAT elements. To investigate the role of Stat6 in regulating IL-4 transcription, we used Stat6-deficient Jurkat T cells with different intact IL-4 promoter constructs in cotransfection assays. We show that, whereas a multimerized response element from the germline IgE promoter was highly induced by IL-4 in Stat6-expressing Jurkat cells, the intact human IL-4 promoter was repressed under similar conditions. We conclude that the function of Stat6 is highly dependent on promoter context and that this factor promotes IL-4 gene expression in an indirect manner.

Binding Sites↗

PubMed1998

Synergistic activation of the germline epsilon promoter mediated by Stat6 and C/EBP beta.

Transcription of the Ig H chain germline transcripts is a prerequisite for class switching. Expression of the epsilon germline transcript is induced by IL-4 and requires the integrity of a composite IL-4 response element. The element is bound by the IL-4-inducible transcription factor Stat6 and one or more members of the CAAT/enhancer-binding protein (C/EBP) family, a constitutively expressed class of transcription factors. Here, we show that Stat6 and C/EBP beta cooperate to synergistically activate transcription from the epsilon element. The effect was most pronounced in lymphoid cells, and the activation domains of both proteins were required to achieve this synergy. Although other members of the C/EBP family are able to bind the element, very little cooperativity was seen with C/EBP alpha and none with C/EBP gamma. In fact, C/EBP gamma was able to inhibit IL-4-induced reporter activity. Stat6 and C/EBP beta bind the IL-4 response element simultaneously. The fast dissociation rate apparent when Stat6 binds this DNA element alone is slowed when C/EBP beta binds at the neighboring site. These data suggest a mechanism whereby C/EBP beta stabilizes Stat6 binding at this element, thereby increasing the likelihood that both of their activation domains will interact, possibly with other factors, to activate transcription in an IL-4-dependent manner.

CCAAT-Enhancer-Binding Proteins↗

PubMed1998

Mutational analysis of the STAT6 SH2 domain.

The SH2 domain of the STAT family of transcription factors is essential for STAT binding to phosphorylated cytoplasmic domains of activated cytokine receptors. Furthermore, the same domain mediates dimerization of activated STAT monomers, a prerequisite for DNA binding by this family of proteins. To identify amino acid residues within the STAT protein that mediate these various interactions, we have carried out an extensive mutational analysis of the Stat6 SH2 domain. Recombinant proteins carrying C-terminal deletions or double alanine substitutions were expressed in mammalian and insect cells and assayed for DNA binding, transcription activation, tyrosine phosphorylation, and the ability to interact with a tyrosine-phosphorylated peptide derived from the interleukin-4 receptor signaling chain. From these studies, we have identified amino acids that are required for both DNA binding and interleukin-4 receptor interaction, as well as residues that when mutated impair only one of the two functions. Our results suggest that the structural homology between the SH2 domain of Stat6 and that of the distantly related Src protein may be higher than predicted on the basis of primary amino acid sequence comparisons. However, the two types of SH2 domains may differ at their C-terminal ends.

Alanine↗

PubMed1998

STAT structure and function in signaling.

The phenotypes of various STAT knockout mice reveal an unexpected specificity in the biological roles of these molecules. The mechanisms involved in generating selectivity and modulating STAT activity have been the focus of intense studies. This work has led to the discovery of novel families of proteins that regulate Jak-STAT signaling. Recently, the structures of a STAT dimer/DNA complex and of the amino-terminal domain have been solved, providing new insights into the function of these versatile proteins.

Animals↗

PubMed1998

Stat proteins control lymphocyte proliferation by regulating p27Kip1 expression.

The proliferation of lymphocytes in response to cytokine stimulation is essential for a variety of immune responses. Recent studies with signal transducer and activator of transcription 6 (Stat6)-deficient mice have demonstrated that this protein is required for the normal proliferation of lymphocytes in response to interleukin-4 (IL-4). In this report, we show that the impaired IL-4-induced proliferative response of Stat6-deficient lymphocytes is not due to an inability to activate alternate signaling pathways, such as those involving insulin receptor substrates, or to a failure to upregulate IL-4 receptor levels. Cell cycle analysis showed that the percentage of Stat6-deficient lymphocytes that transit from the G1 to the S phase of the cell cycle following IL-4 stimulation is lower than that of control lymphocytes. Although the regulation of many genes involved in the control of cytokine-induced proliferation is normal in Stat6-deficient lymphocytes, protein levels of the cdk inhibitor p27Kip1 were found to be markedly dysregulated. p27Kip1 is expressed at significantly higher levels in Stat6-deficient lymphocytes than in control cells following IL-4 stimulation. The higher level of p27Kip1 expression seen in IL-4-stimulated Stat6-deficient lymphocytes correlates with decreased cdk2-associated kinase activity and is the result of the increased accumulation of protein rather than altered mRNA expression. Similarly, higher levels of p27Kip1 protein expression are also seen following IL-12 stimulation of Stat4-deficient lymphocytes than are seen following stimulation of control cells. These data suggest that Stat proteins may control the cytokine-induced proliferative response of activated T cells by regulating the expression of cell cycle inhibitors so that cyclin-cdk complexes may function to promote transition from the G1 to the S phase of the cell cycle.

Animals↗

PubMed1997

Interaction of STAT5 dimers on two low affinity binding sites mediates interleukin 2 (IL-2) stimulation of IL-2 receptor alpha gene transcription.

Stimulation of the interleukin 2 receptor alpha (IL-2Ralpha) gene by IL-2 is important for the proliferation of antigen-activated T lymphocytes. IL-2 regulates IL-2Ralpha transcription via a conserved 51-nucleotide IL-2 responsive enhancer. Mouse enhancer function depends on cooperative activity of three distinct sites. Two of these are weak binding sites for IL-2-activated STAT5 (signal transducer and activator of transcription) proteins, and mutational analysis indicates that binding of STAT5 to both sites is required for IL-2 responsiveness of the enhancer. The STAT5 dimers interact to form a STAT5 tetramer. The efficiency of tetramerization depends on the relative rotational orientation of the two STAT motifs on the DNA helix. STAT5 tetramerization on enhancer mutants correlates well with the IL-2 responsiveness of these mutants. This provides strong evidence that interactions between STAT dimers binding to a pair of weak binding sites play a biological role by controlling the activity of a well characterized, complex cytokine-responsive enhancer.

Animals↗

PubMed1997

A high-throughput STAT binding assay using fluorescence polarization.

STAT (signal transducers and activators of transcription) is a class of transcription factors that are activated upon cytokine or growth factor binding to cell surface receptors. Activated STAT proteins dimerize, translocate into the nucleus, and activate transcription, leading to various immune responses. The inhibition of STAT binding to cell receptors might provide a means to modulate these immune responses. We developed a high-throughput biochemical assay to measure the interaction between an interferon-gamma receptor-derived phosphotyrosine-containing peptide and STAT1, using fluorescence polarization as the detection method. This assay can be used to screen for small molecules capable of disrupting receptor-STAT interactions.

Binding, Competitive↗

PubMed1996

No .NO from NO synthase.

The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of L-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form L-citrulline (Cit) and nitric oxide (.NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify .NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable .NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal .NO standards were only partially recovered and the dismutation of superoxide (O2-.), which otherwise scavenges. .NO to yield ONOO-, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO- resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8-1.1, suggesting either that additional reducing equivalents besides NADPH are required to explain Arg oxidation to .NO or that .NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H4Bip. Here, recovery of internal .NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO-) to .NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from .NO decomposition, was consistent with formation of an .NO precursor molecule such as NO-. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that .NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.

Electron Transport↗

PubMed1996

Stat6 is required for mediating responses to IL-4 and for development of Th2 cells.

Interleukin-4 (IL-4) stimulation of cells leads to the activation of multiple signaling pathways, one of which involves Stat6. We have generated Stat6-deficient mice by gene targeting in embryonic stem cells to determine the role of this transcription factor in mediating the biologic functions of IL-4. IL-4-induced increases in the cell surface expression of both MHC class II antigens and IL-4 receptor are completely abrogated, and lymphocytes from Stat6-deficient animals fail to proliferate in response to IL-4. Stat6-deficient B cells do not produce IgE following in vivo immunization with anti-IgD. In addition, Stat6-deficient T lymphocytes fail to differentiate into Th2 cells in response to either IL-4 or Il-13. These results demonstrate that, despite the existence of multiple signaling pathways activated by IL-4, Stat6 is essential for mediating responses to IL-4 lymphocytes.

Animals↗

PubMed1996

Differentiation of T-helper lymphocytes: selective regulation by members of the STAT family of transcription factors.

Interleukin-4 (IL-4) and interleukin-12 (IL-12) control the differentiation of T-helper cells. Here we summarize studies which investigate the mechanism by which these cytokines selectively reprogramme gene expression in T-lymphocytes. Cytokine stimulation leads to the phosphorylation of specific tyrosine residues within the intracellular domain of the corresponding cytokine receptor. These phosphotyrosines serve as docking sites for latent, cytoplasmic transcription factors known as signal transducers and activators of transcription (Stat) proteins. Receptor/Stat interaction is mediated by the src homology 2 (SH2) domain of the corresponding Stat protein. Although Stat binding to the intracellular domain of the cytokine receptor strongly depends on the phosphotyrosine residue, the recruitment of a specific Stat protein is dictated by amino acid residues C-terminal to the phosphotyrosine. Specific docking sites within individual cytokine receptors have been identified for almost all Stat proteins. The direct coupling between cytokine receptor and transcription factor helps to explain how different cytokines elicit distinct patterns of gene expression.

Animals↗

PubMed1996

Requirements for interleukin-4-induced gene expression and functional characterization of Stat6.

Interleukin-4 (IL-4) stimulation leads to the activation of the signal transducer and activator of transcription 6 (Stat6). In this study, we present data relating to the functional properties of Stat6. Human embryonic kidney 293 cells were shown to be deficient of Stat6 yet express all other components of the IL-4 signaling cascade. This cell line was used for transient-transfection studies of wild-type and mutant Stat6 proteins. The wild-type protein was shown to activate a reporter construct carrying multiple copies of the IL-4 response element derived from the human immunoglobulin heavy-chain germ line epsilon promoter. Similarly, a truncated protein lacking 41 amino acids of the N terminus was fully active. However, removal of the C-terminal 186 amino acids completely abolished transcription activation. Amino acid substitutions were introduced into the putative DNA binding domain (VVI at positions 411 to 413), the SH2 domain (R-562), or the tyrosine (Y-641) which presumably becomes phosphorylated upon activation. All three of these Stat6 mutants were unable to activate transcription in 293 cells. Wild-type and mutant Stat6 derivatives were also expressed in insect cells, and purified proteins were analyzed in vitro for the ability to interact with both DNA and tyrosine-phosphorylated peptides derived from the IL-4 receptor alpha chain. Mutations within the DNA binding domain, the SH2 domain, or tyrosine 641 completely abolished DNA binding. In contrast, only the SH2 mutant failed to interact with tyrosine-phosphorylated peptides. The transdominant effects of all Stat6 derivatives were analyzed by using HepG2 cells, which express endogenous Stat6 protein. Differential effects were observed with various mutants, supporting the current model of the Jak/STAT activation cycle.

Amino Acid Sequence↗

PubMed1995

Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta chain but is not essential for the proliferative signal transmission.

The high-affinity interleukin 2 (IL-2) receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two members of the Janus kinase family, Jak1 and Jak3, are associated with IL-2R beta c and IL-2R gamma c, respectively, and they are activated upon IL-2 stimulation. The cytokine-mediated Jak kinase activation usually results in the activation of a family of latent transcription factors termed Stat (signal transducer and activator of transcription) proteins. Recently, the IL-2-induced Stat protein was purified from human lymphocytes and found to be the homologue of sheep Stat5/mammary gland factor. We demonstrate that the human Stat5 is activated by IL-2 and that Jak3 is required for the efficient activation. The cytoplasmic region of the IL-2R beta c chain required for activation of Stat5 is mapped within the carboxyl-terminal 147 amino acids. On the other hand, this region is not essential for IL-2-induced cell proliferation.

3T3 Cells↗

PubMed1995

Components of a Stat recognition code: evidence for two layers of molecular selectivity.

Latent and activated forms of Stat1 and Stat6 have been expressed and purified, enabling biochemical experiments relating to their functional activities. Stat1 bound to a phosphotyrosine peptide derived from the IFN gamma receptor with a KD of 50 nM, whereas Stat6 bound to an IL-4 receptor peptide with a KD of 300 nM. Stat-receptor peptide interactions were specific and dependent upon tyrosine phosphorylation. Activated forms of Stat1 and Stat6 were used to select their optimal DNA binding sites. Stat1 selected a recognition site having dyad half-sites separated by 3 bp. Stat6 selected a recognition site composed of the same dyad half-sites, yet separated by 4 bp. Chimeric Stat1-Stat6 recombinants were expressed, purified, and assayed for receptor coupling and DNA binding specificity. Such studies led to the identification of polypeptide domains that specify these activities. These observations provide a framework for understanding how different cytokines elicit distinctive patterns of gene expression.

Amino Acid Sequence↗

PubMed1995

Identification and purification of human Stat proteins activated in response to interleukin-2.

A key cytokine induced during the immune response is IL-2. Following T cell activation, the genes encoding IL-2 and the various chains of its receptor are transcriptionally induced. In turn, secreted IL-2 serves to stimulate the proliferation and differentiation of T lymphocytes. Several recent studies have implicated Jak kinases in the signaling pathway induced by IL-2. Following this lead, we set out to identify transcription factors induced in response to IL-2. Human peripheral blood lymphocytes were observed to contain several IL-2-inducible DNA binding activities. Similar activities were also observed in a transformed human lymphocyte line, termed YT. We have purified these activities and found that the principal IL-2-inducible component bears significant relatedness to a prolactin-induced transcription factor first identified in sheep mammary gland tissue. We hypothesize that activation of this protein, designated hStat5, helps govern the biological effects of IL-2 during the immune response.

Amino Acid Sequence↗

PubMed1995

The G-box: a ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins.

The G-box (CACGTG) is a ubiquitous, cis-acting DNA regulatory element found in plant genomes. Proteins known as G-box factors (GBFs) bind to G-boxes in a context-specific manner, mediating a wide variety of gene expression patterns. We suggest that, as for many biological systems, different combinations of these common elements can lead to diversity and specificity in the regulation of plant gene expression.

Amino Acid Sequence↗

PubMed1995

Detection of dihydrotestosterone (DHT) doping: alterations in the steroid profile and reference ranges for DHT and its 5 alpha-metabolites.

Dihydrotestosterone (DHT), a biologically active metabolite of testosterone, may be misused in sports to benefit from its anabolic and psychotropic effects. After DHT application, a significant increase of the glucuronides of DHT and its metabolites can be expected for a certain time period depending upon dose, formulation, route of administration, and in case of percutaneous administration the chainlength of the ester. DHT and its metabolites can be monitored by gas-chromatography/mass spectrometry (GC/MS) after enzymatic hydrolysis and trimethylsilylation. To investigate the extent of the alteration of the urinary steroid profile after DHT application, timely controlled experiments have been performed with: a) oral application of [16,16,17-2H3]-DHT, and b) sublingual application of a 25 mg dose of DHT. In the experiment with [16,16,17-2H3]-DHT within 24 hours about 44% of the applied dose was recovered after hydrolysis with beta-glucuronidase from E. coli as di- or tri-deuterated 5 alpha-androstane glucuronides: androsterone (33.2%), 5 alpha-androsta-ne-3 alpha,17 beta-diol (2.5%), 5 alpha-androstane-3 beta, 17 beta-diol (0.9%), DHT (7.2%). Hydrolysis with beta-glucuronidase/arylsulfatase from Helix Pomatia resulted in a about 10% higher yield except for DHT. In the study with sublingual application of 25 mg of DHT the extent of the recovery of DHT and its metabolites was in the same range as for the deuterated DHT. The urinary glucuronide concentrations of DHT, androsterone (AND), 5 alpha-androstane-3 alpha, 17 beta-diol (5 alpha A3 alpha D) and 5 alpha-androstane-3 beta,17 beta-diol (5 alpha A3 beta D) and their ratios to etiocholanolone (ETIO), 5 beta-androstane-3 alpha, 17 beta-diol (5 beta A3 alpha D) and epitestosterone (EPIT) were increased for up to 48 hours after application. For doping control purposes concentrations of DHT, 5 alpha A3 alpha D, 5 alpha A3 beta D and ratios of 5 alpha-metabolites to non 5 alpha-metabolites such as DHT/ETIO, DHT/EPIT, 5 alpha A3 alpha D/5 beta A3 alpha D, 5 alpha A3 beta D/5 beta A3 alpha D, and AND/ETIO outside the reference ranges are a proof for DHT application. Reference ranges for Asian and Caucasian male and female athletes are calculated from data bases of the Asian Games 1994, the previous Asian Games 1990 and the routine doping control samples of Caucasian athletes measured in Cologne 1994. At the occasion of the 1994 Asian Games in Hiroshima alterations in the concentrations and ratios of the DHT depending parameters for outside there reference ranges have been found and have been sanctioned on this basis by the Medical Commission of the Organisation of Olympic Council of Asia (OCA).

Adult↗

PubMed1994

An interleukin-4-induced transcription factor: IL-4 Stat.

Interleukin-4 (IL-4) is an immunomodulatory cytokine secreted by activated T lymphocytes, basophils, and mast cells. It plays an important role in modulating the balance of T helper (Th) cell subsets, favoring expansion of the Th2 lineage relative to Th1. Imbalance of these T lymphocyte subsets has been implicated in immunological diseases including allergy, inflammation, and autoimmune disease. IL-4 may mediate its biological effects, at least in part, by activating a tyrosine-phosphorylated DNA binding protein. This protein has now been purified and its encoding gene cloned. Examination of the primary amino acid sequence of this protein indicates that it is a member of the signal transducers and activators of transcription (Stat) family of DNA binding proteins, hereby designated IL-4 Stat. Study of the inhibitory activities of phosphotyrosine-containing peptides derived from the intracellular domain of the IL-4 receptor provided evidence for direct coupling of receptor and transcription factor during the IL-4 Stat activation cycle. Such observations indicate that IL-4 Stat has the same functional domain for both receptor coupling and dimerization.

Amino Acid Sequence↗

PubMed1994

The cognition-enhancing drug tenilsetam is an inhibitor of protein crosslinking by advanced glycosylation.

Non-enzymatic glycosylation of proteins, also called Maillard reaction, which occurs at an accelerated rate in diabetes, can lead to the formation of advanced glycosylation endproducts (AGEs). Tenilsetam (CAS 997: (+/-)-3-(2-thienyl)-2-piperazinone), a cognition-enhancing drug successfully used for treatment of patients suffering from Alzheimer's disease, when included in the Maillard reaction apparently inhibits protein crosslinking by AGEs in vitro. According to the mechanism proposed, Tenilsetam acts via covalent attachment to glycated proteins, thus blocking the reactive sites for further polymerisation reactions. A beneficial effect of Tenilsetam in Alzheimer's disease could come from the interference with AGE-derived crosslinking of amyloid plaques and a decreased inflammatory response by diminished activation of phagocytosing microglia.

Aging↗